Synthesis of 2′‐Cyclohexenylnucleosides and Corresponding CeNA Building Blocks

Feng‐Wu Liu1, Alberto Di Salvo1, Piet Herdewijn1

1 Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, Leuven, Belgium
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.20
DOI:  10.1002/0471142700.nc0120s33
Online Posting Date:  June, 2008
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Abstract

A cyclohexene ring has similar structural properties and conformational behavior to a saturated five‐membered furanose ring. In particular, it has good hydrolytic stability. Cyclohexenylnucleosides have been utilized in antiviral drug design, and some nucleosides and oligonucleotides based on the cyclohexene system have been developed. For further investigation of these modified nucleosides and oligonucleotides, synthesis of the chiral cyclohexenylnucleosides in high enantiomeric excess and in bulk quantities is necessary. This unit describes the complete synthesis of four enantiomerically pure 5′‐hydroxy‐4′‐hydroxymethyl‐2′‐cyclohexenylnucleosides (thymine, cytosine, guanine, and adenine) and the four corresponding N‐protected 4′‐(monomethoxytrityl)oxymethyl cyclohexenyl nucleic acids (CeNA) building blocks. The chirality of these compounds is 1′S, 4′R, and 5′S. Curr. Protoc. Nucleic Acid Chem. 33:1.20.1‐1.20.21. © 2008 by John Wiley & Sons, Inc.

Keywords: cyclohexenylnucleosides; Diels‐Alder cycloaddition; enzymatic resolution; Mitsunobu reaction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Enantiomerically Pure Cyclohexenyl Precursor
  • Basic Protocol 2: Preparation of 2′‐Cyclohexenylthymine Derivatives
  • Basic Protocol 3: Preparation of 2′‐Cyclohexenylcytosine Derivatives
  • Basic Protocol 4: Preparation of 2′‐Cyclohexenylguanine Derivatives
  • Basic Protocol 5: Preparation of 2′‐Cyclohexenyladenine Derivatives
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Enantiomerically Pure Cyclohexenyl Precursor

  Materials
  • Ethyl acetate, analytical grade
  • Ethyl formate, analytical grade
  • Sodium pieces
  • Diethyl ether, anhydrous
  • Acetyl chloride (AcCl)
  • Saturated aqueous sodium bicarbonate
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel for column chromatography (60Å, 63 to 200 µm)
  • n‐Hexane
  • Dichloromethane (CH 2Cl 2), reagent grade
  • Zinc chloride, anhydrous
  • Triethylamine, reagent grade
  • (E)‐4‐Methoxy‐3‐buten‐2‐one (purity 90%)
  • Toluene, analytical grade
  • Chlorotrimethylsilane (TMSCl)
  • Nitrogen balloon
  • Celite
  • Hydroquinone, reagent grade
  • Tetrahydrofuran (THF), anhydrous and reagent grade
  • Lithium aluminum hydride (LiAlH 4)
  • 15% NaOH
  • Methanol (MeOH), reagent grade
  • Ethyl acetate (EtOAc)
  • Benzaldehyde dimethyl acetal
  • Dioxane, reagent grade
  • p‐Toluenesulfonic acid monohydrate
  • Manganese(IV) dioxide (MnO 2), precipitated active
  • Cerium(III) chloride heptahydrate (CeCl 3·7H 2O)
  • Sodium borohydride (NaBH 4)
  • Isopropenyl acetate
  • Novozyme 435 (Sigma)
  • 100‐, 500‐, and 1000‐mL round‐bottom flasks
  • Magnetic stir bar and stir plate
  • Rubber stopper
  • 0.9 ×40–mm needles
  • Room‐temperature and 40°C water baths
  • Dropping funnels
  • 1000‐mL separatory funnels
  • Rotary evaporator
  • Vacuum pump
  • 4.5 × 45–, 5 × 50–, and 6 × 60–cm gel columns
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Glass filters
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of 2′‐Cyclohexenylthymine Derivatives

  Materials
  • Thymine, reagent grade
  • Pyridine, reagent grade
  • Benzoyl chloride
  • Sodium bicarbonate, saturated, aqueous
  • Ethyl acetate (EtOAc)
  • Dioxane
  • Potassium carbonate (K 2CO 3)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • (4aR,7R,8aS)‐2‐Phenyl‐4a,7,8,8a‐tetrahydro‐4H‐benzo[d][1,3]dioxin‐7‐ol (S.4)
  • Sodium benzoate
  • Triphenylphosphine (Ph 3P)
  • Tetrahydrofuran (THF), anhydrous
  • Diisopropyl azodicarboxylate (DIAD)
  • Nitrogen balloon
  • Celite
  • Saturated ammonia (NH 3) in methanol (MeOH), saturated at 0°C
  • Methanol (MeOH), reagent grade
  • Trifluoroacetic acid
  • Toluene
  • Silica gel for column chromatography (60 Å, 63 to 200 µm)
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Dichloromethane (CH 2Cl 2)
  • 4‐Monomethoxytrityl chloride (MMTrCl)
  • 4‐Dimethylaminopyridine (DMAP)
  • 100‐, 250‐, and 1000‐mL round‐bottom flasks
  • Magnetic stir bars and plate
  • Dropping funnels
  • Rotary evaporator connected to a vacuum pump
  • Glass filters
  • 1.5 × 28– and 2.5 × 40–cm gel columns
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of 2′‐Cyclohexenylcytosine Derivatives

  Materials
  • Uracil, reagent grade
  • (4aR,7R,8aS)‐2‐Phenyl‐4a,7,8,8a‐tetrahydro‐4H‐benzo[d][1,3]dioxin‐7‐ol (S.4)
  • Sodium benzoate
  • Triphenylphosphine (Ph 3P)
  • Tetrahydrofuran (THF dried over Na)
  • 40% diethyl azodicarboxylate (DEAD) solution in toluene
  • Nitrogen balloon
  • Saturated ammonia in methanol (NH 3 in MeOH), saturated at 0°C
  • Methanol (MeOH), reagent grade
  • Silica gel for column chromatography (60 Å, 63 to 200 µm)
  • Ethyl acetate (EtOAc)
  • n‐Hexane
  • 1,2,4‐Triazole
  • Pyridine, anhydrous
  • Phosphorus(V) oxychloride (POCl 3)
  • Dioxane
  • 28% ammonium hydroxide solution, reagent grade
  • Dichloromethane (CH 2Cl 2)
  • Trifluoroacetic acid (TFA)
  • Toluene
  • Benzoyl chloride (BzCl)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • 4‐Monomethoxytrityl chloride (MMTrCl)
  • 4‐Dimethylaminopyridine (DMAP)
  • 50‐, 100‐, and 1000‐ml round‐bottom flasks
  • 250‐mL dropping funnel
  • Glass filters
  • Rotary evaporator connected to a vacuum pump
  • 1.5 × 28– and 2.5 × 40–cm gel columns
  • TLC plates: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Preparation of 2′‐Cyclohexenylguanine Derivatives

  Materials
  • (4aR,7R,8aS)‐2‐Phenyl‐4a,7,8,8a‐tetrahydro‐4H‐benzo[d][1,3]dioxin‐7‐ol (S.4)
  • 2‐Amino‐6‐chloropurine (S.19)
  • Triphenylphosphine (Ph 3P)
  • Tetrahydrofuran (THF; dried over Na)
  • 40% diethyl azodicarboxylate (DEAD) solution in toluene
  • Nitrogen balloon
  • Celite
  • Silica gel for column chromatography (60 Å, 63 to 200 µm)
  • Ethyl acetate (EtOAc)
  • n‐Hexane
  • Trifluoroacetic acid (TFA)
  • Toluene
  • Methanol (MeOH), reagent grade
  • Dichloromethane (CH 2Cl 2)
  • Pyridine (dried over KOH)
  • Chlorotrimethylsilane (TMSCl)
  • Isobutyryl anhydride
  • 4‐Monomethoxytrityl chloride (MMTrCl)
  • 4‐Dimethylaminopyridine (DMAP)
  • 100‐ and 250‐mL round‐bottom flasks
  • 50‐mL dropping funnels
  • Rotary evaporator connected to a vacuum pump
  • 2.5 × 40– and 4.5 × 47–cm gel columns
  • TLC plates: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 5: Preparation of 2′‐Cyclohexenyladenine Derivatives

  Materials
  • (4aR,7R,8aS)‐2‐Phenyl‐4a,7,8,8a‐tetrahydro‐4H‐benzo[d][1,3]dioxin‐7‐ol (S.4)
  • Adenine (S.24)
  • Dioxane (dried over Na)
  • Diisopropyl azodicarboxylate (DIAD)
  • Nitrogen gas
  • Silica gel for column chromatography (60 Å, 63 to 200 µm)
  • Ethyl acetate (EtOAc)
  • Dichloromethane (CH 2Cl 2)
  • Methanol (MeOH), reagent grade
  • 80% aqueous acetic acid
  • Toluene
  • Ethanol
  • Pyridine (dried over KOH)
  • Chlorotrimethylsilane (TMSCl)
  • Benzoyl chloride (BzCl)
  • 28% ammonium hydroxide
  • Hexane
  • 4‐Monomethoxytrityl chloride (MMTrCl)
  • Triethylamine (Et 3N)
  • 50‐ and 100‐ml round‐bottom flasks
  • Magnetic stir bars and plate
  • Dropping funnel
  • 1.5 × 28–cm chromatography columns
  • TLC plates: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Rotary evaporator connected to a vacuum pump
  • Preparative TLC (pTLC) plates (20 × 40–cm glass plates coated with MN‐silica gel P/UV 254)
  • Filter paper
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Gu, P., Griebel, C., Van Aerschot, A., Rozenski, J., Busson, R., Gais, H.‐J., and Herdewijn, P. 2004. Synthesis of enantiomeric‐pure cyclohexenyl nucleoside building blocks for oligonucleotide synthesis. Tetrahedron 60:2111‐2123.
   Herdewijn, P. 1999. Conformationally restricted carbohydrate‐modified nucleic acids and antisense technology. Biochim. Biophys. Acta 1489:167‐179.
   Maurinsh, Y., Rosemeyer, H., Esnouf, R., Medvedovici, A., Wang, J., Ceulemans, G., Lescrinier, E., Hendrix, C., Busson, R., Sandra, P., Seela, F., Van Aerschot, A., and Herdewijn, P. 1999. Synthesis and pairing properties of oligonucleotides containing 3‐hydroxy‐4‐hydroxymethyl‐1‐cyclohexanyl nucleosides. Chem. Eur. J. 5:2139‐2150.
   Nauwelaerts, K., Fisher, M., Froeyen, M., Lescrinier, E., Van Aerschot, A., Xu, D.G., DeLong, R., Kang, H., Juliano, R.L., and Herdewijn, P. 2007. Incorporation of a single cyclohexenyl nucleic acid (CeNA) unit in the sense and antisense of siRNA increases biological activity. J. Am. Chem. Soc. 129:9340‐9348.
   Vastmans, K., Pochet, S., Peys, A., Kerremans, L., Van Aerschot, A., Hendrix, C., Marlière, P., and Herdewijn, P. 2000. Enzymatic incorporation in DNA of 1,5‐anhydrohexitol nucleotides. Biochemistry 39:12757‐12765.
   Verbeure, B., Lescrinier, E., Wang, J., and Herdewijn, P. 2001. RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA). Nucleic Acids Res. 29:4941‐4947.
   Wang, J. and Herdewijn, P. 1999. Enantioselective synthesis and conformational study of cyclohexene carbocyclic nucleosides. J. Org. Chem. 64:7820‐7827.
   Wang, J., Froeyen, M., Hendrix, C., Andrei, G., Snoeck, R., De Clercq, E., and Herdewijn, P. 2000a. The cyclohexene ring system as a furanose mimic: Synthesis and antiviral activity of both enantiomers of cyclohexenylguanine. J. Med. Chem. 43:736‐745.
   Wang, J., Verbeure, B., Luyten, I., Lescrinier, E., Froeyen, M., Hendrix, C., Rosemeyer, H., Seela, F., Van Aerschot, A., and Herdewijn, P. 2000b. Cyclohexene nucleic acids (CeNA): Serum stable oligonucleotides that activate RNase H and increase duplex stability with complementary RNA. J. Am. Chem. Soc. 122:8595‐8602.
   Wang, J., Morral, J., Hendrix, C., and Herdewijn, P. 2001. A straightforward stereoselective synthesis of d‐ and l‐5‐hydroxy‐4‐hydroxymethyl‐2‐cyclohexenylguanine. J. Org. Chem. 66:8478‐8482.
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